1. Field of the Invention
This invention relates in general to a method of producing a cloned chitinase enzyme, as well as to a cloned chitinase enzyme from Vibrio parahemolyticus.
2. Prior Art
Chitin is a polymer of beta 1-4 linked 2-dexoy-2-acetamidoglucopyranose (poly-N-acteylglucosamine) which is one of the largest sources of renewable biomass on earth, second only to cellulose and lignins. For example, chitin forms the exo-skeleton of insects and crustaceans and is a component of cell walls in some fungi and yeasts.
The first and most important step in the natural breakdown of this substance occurs when microbes and fungi secrete the enzyme chitinase which degrades the largely insoluble polymer to di-N-acteyl chitobiose. A usual second step involves the enzyme chitobiase which degrades the soluble chitobiose to N-acteylglucosamine. This substance, since it contains bound nitrogen and, as a sugar, is a carbon source, could be utilized for many fermentation processes such as a valuable feed stock for obtaining single cell protein, or as a feed supplement for pisci-culture, chickens, pigs or cattle.
Sources of chitin that are now principally considered a waste by-product would include shellfish shells. More particularly, crayfish, shrimp and crab shells, which consists mainly of chitin, protein and calcium carbonate would make excel lent sources or chitin, since approximately 25-58% of the shellfish waste is chitin.
Unfortunately, the industrial use of the degradation products chitobiose and N-acetyl glucosamine have been restricted by the expense and unavailability of these substances. Much of the research performed thus far on commercial use of chitin in shellfish waste has taken place since 1957. L. R. Berger studied digestion of chitin by an enzyme called chitinase from a streptomyces species. "Digestion of chitin by a Streptomyces Species", L. R. Berger, Ph.D. Thesis, University of California, Davis, Calif. In 1970 J. G. Chan studied chitin degrading organisms in Puget Sound and reported his findings in "The Occurrence, Taxonomy, and Activity of Chitinoclastic Bacteria from Sediment, Water, and Fauna of Puget Sound", J. G. Chart, Ph.D. Thesis, University of Washington, Seattle, Wash. The United States Environmental Protection Agency in 1971 noted concerns about pollution caused by shellfish waste. "Pollution Abatement and By-product Recovery in Shellfish and Fisheries Processing", Project # 12130FJQ, Environmental Protection Agency, Washington, D.C. In 1975, B. L. Averback investigated the structure of chitin and chitosan. "The Structure of Chitin and Chitosan, M.I.T. Sea Grant Program Report # MITSG77E, Index #77-703Z1e, Massachusetts Institute of Technology, Cambridge, Mass. Then, in 1977, N. A. Ashford, et al. reported on industrial prospects for chitin and protein recovery from shellfish waste. "Industrial Prospects for Chitin and Protein from Shellfish Wastes", M.I.T. Sea Grant Program #MITSG-77-3, Index #77-703-Z1e, Massachusetts Institute of Technology, Cambridge, Mass. A year later, P. A. Carroad and R. A. Tom proposed a process conception and selection of microorganisms for bioconversion of shellfish chitin wastes. "Bioconversion of Shellfish Chitin Wastes: Process Conception and Selection of Micro-organisms, Journal of Food Science, Vol. 43, pages 1158-1161. They singled out a Serratia marcescens and a Bacillus cereus as the most promising chitinase enzyme producers for exploitation. D. M. Ogrydziak and J. D. Reid, in 1980, described an isolate of Serratia marcescens as a mutant overproducer of the enzyme chitinase. "Chitinase-Overproducing Mutant of Serratia Marcescens" Applied & Environmental Microbiology, Vol. 41, page 664. In 1981, R. A. Tom and P. A. Carroad reported on the effects of reaction conditions on hydrolysis of chitin by the enzyme from Ogrydziak's mutant Serratia marcescens. "Effect of Reaction Conditions on Hydrolysis of Chitin by Serratia marcescens QMB 1466 Chitinase," Journal of Food Science 46, pages 646-647. I. G. Casio et al. proposed in 1982 a scheme for bioconversion of chitin from shrimp shell waste with several acid and base pretreatments, followed by chitinase from Serratia marcescens. "Bioconversion of Shellfish Chitin Waste: Waste Pretreatment, Enzyme Production, Process Design, and Economic Analysis," Journal of Food Science 47, pages 901-905. They suggested a process design and performed an economic analysis that indicated a negative after-tax cash flow of $0.06/kg. However, several of the expensive factors in the economic analysis of biodegradative processing of shrimp shell waste used in performing the economic analysis could be mitigated by today's technology. In 1986, two chitinase genes from Serratia marcescens QMB1466, have been reported by R. L. Fuchs, S. A. McPherson and D. J. Drahos in "Cloning of a Serratia marcescens Gene Encoding Chitinase", Applied Environmental Microbiology 51, 504-509 and by J. D. G. Jones, K. L. Grady, T. V. Suslow and J. R. Bedbrook in "Isolation and Characterization of Genes Encoding Two Chitinase Enzymes from Serratia marcescens", EMBO 5, 467-473, to have been cloned in E. coli and the nucleotide sequence of one of these genes determined.
However, the physical stability of the chitinase from Serratia marcescens is not as great as would be desired. Furthermore, accessibility of the substrate, chitin, by the enzyme is limited by the presence of proteins and calcium carbonate, which are, by current technology, removed by treatment with a base, and an acid, respectively. A chitinase enzyme to be used in the latter system would be much better if it is salt-tolerant, since, after neutralization of the acid or base treatment, perhaps 1M salt would be present. A process step for removal of the salt prior to digestion is a time-consuming and expensive step. For this reason the more stable chitinase enzyme from Vibrio parahemolyticus, a halophilic microorganism which requires 2-7% sodium chloride for growth, could possibly serve as a better enzyme for digestion of chitin from shellfish, than those from Serratia marcescens and Streptomyces griseus. Chitinases isolated from an unidentified species of Vibrio have been reported in 1979 by Y. Uchida, M. Misutome and A. Ohitakara in "Isolation of a Chitinase EC-3.2.1.14 Producing Bacterium: Culture Conditions for Enzyme Production and Properties of the Enzymes," Japan Fermentation Technology 57, 131-140, as well as in "Purification and Some Properties of Chitinase EC-3.2.1.14 from Vibrio Species," Japan Fermentation Technology 57, 169-177.